(A) rKSHV-EA.hy926 cells (rKSHV-Ea.hy) were transfected with siRNA oligonucleotides specific for Pim-1, -2, or -3, or with control siRNA (si ctrl), and subjected to maximal reactivation (RTA+NaB) 48 h after transfection. After another 48 h, whole cell extracts were collected and analyzed by Western blotting with anti-Pim-1, -2, -3, and -K8.1 antibodies. Quantification of K8.1 protein level is shown below the panel. Anti-tubulin served as a loading control. (B) Cells were treated as in (A), and 30 h after maximal reactivation, the cells were analyzed for RFP expression. 72 h after reactivation, supernatants were collected and used to infect naive U2OS cells. (C) 72 h after infection, the U2OS cells were analyzed for GFP expression (a measure of production of infectious virions). Values are means of two independent experiments ±SD.

(A) 293 cells were transfected with V5-tagged expression constructs for either wt or kinase-deficient (KD) Pim-1 or -3. After 48 h, cell extracts were immunoprecipitated by anti-V5 antibodies and subjected for in vitro kinase assay on GST-C-LANA or GST-N-LANA. The samples were resolved by SDS-PAGE (8%), and followed by autoradiography. The kinase filter was blotted with anti-GST antibody and anti-V5 antibodies. (B) The truncated fragments of the LANA N-terminus are illustrated in the upper part of the panel. 293 cells were transfected with V5-tagged expression constructs for either Pim-1 or -3. After 48 h, the in vitro kinase assay was performed essentially as described in (A) on GST-fusions of N-terminal truncations of LANA as indicated. Coomassie staining was used to detect the substrates. (C) 293 cells were transfected with wt (LANA wt) or two different clones of the SS205/206RR mutant of LANA (SS205/206RR (1), and SS205/206RR (2)) with and without V5-tagged expression constructs for Pim-1 or -3 as indicated. After 48 h, cell extracts were collected and subjected to an in vitro kinase assay essentially as described in (A) on co-precipitated proteins. The samples were resolved by SDS-PAGE (8%) followed by autoradiography. The kinase filter was immunoblotted with anti–LANA, and anti-V5 antibodies to ensure the efficiency of immunoprecipitation. An arrow indicates phosphorylated LANA band on the right. (D) BC-3 cells were treated with TPA for 48 h. Whole cell extracts were incubated with GST-N-LANA and GST-C-LANA alone or in combination for 6 hrs and subjected to immunoprecipitation with anti-Pim-1 antibody, followed by in vitro kinase assay on co-precipitated proteins. GST alone (GST) was used as a negative control. The kinase filter was immunoblotted with anti–LANA, –GST, and –Pim-1 antibodies. The arrows indicate phosphorylated LANA and GST-N-LANA on the right. (E) Recombinant GST-fusion proteins (GST, GST-N-LANA, or GST-C-LANA) were incubated with purified wild-type (wt) or kinase-dead (KD) Pim-1 proteins and subjected to an in vitro kinase assay. Samples were resolved by SDS-PAGE and autoradiographed. The substrates indicated on the right were visualized by Coomassie staining.

BC-3 cells were first synchronized to S (Pim-1 si)-phase and treated with increasing amount of IFN-γ for 3 or 16 h. TPA treatment was used as a positive control. (A) Cell extracts were analyzed for the expression of Pim-1 by Western blot, and (D) for expression of ORF50 and ORF57 lytic transcripts by qRT-PCR. (B) BC-3 and BJAB cells were treated with 1 or 5 U/ml of IFN-γ for 16 h, and whole cell extracts were immunoprecipitated with anti-Pim-1 antibodies and subjected to an in vitro kinase assay on co-precipitated proteins. The samples were resolved by SDS-PAGE (8%) followed by autoradiography. The kinase filter was immunoblotted with anti–LANA and anti–Pim-1 antibodies. Anti-tubulin served as a loading control. (C) BC-3 cells were transfected with siRNAs specific for Pim-1 (Pim-1 si) or control siRNA (ctrl si) or left untreated (−). 48 h after transfection, cells were treated for 16 h with IFN-γ as described above. Cell extracts were analyzed for the silencing of Pim-1 expression by Western blot with anti-Pim-1 antibodies, and (D) for expression of ORF50 and ORF57 lytic transcripts by qRT-PCR. Anti-tubulin served as a loading control in C.

(A) rKSHV.219–infected Vero cells (rKSHV-Vero) or (D) EA.hy926 cells (rKSHV-Ea.hy) were transfected with the V5-tagged expression vectors for wt and kinase-deficient (KD) forms of Pim kinases as indicated and infected with RTA baculovirus 48 h after transfection (negative control). NaB and infection with RTA baculovirus were used to induce maximal reactivation (positive control). Empty vector transfection was used in the negative and positive controls. 30 h later, the cells were analyzed for RFP expression. 96 h after adding RTA or RTA and NaB, the supernatants from KSHV-Vero (B) or KSHV-EA.hy926 (E) cells were collected and used to infect U2OS target cells. 72 h after infection, U2OS cells were analyzed for GFP expression (a measure of production of infectious virions). Values are means of two independent experiments ±SD. 48 h after addition of RTA, cell extracts from rKSHV-Vero (C) or rKSHV-EA.hy926 (F) were analyzed by Western blotting with antibodies against lytic protein K8.1 and the V5-tag on Pim kinases. Anti-tubulin served as a loading control.

(A) BCBL-1, BC-3, as well as a KSHV–negative control cell line BJAB, were treated with TPA (+) or solvent (DMSO; −) for 48 h. Whole cell extracts were immunoblotted with anti-Pim-1, -3, and -K8.1 antibodies. Anti-tubulin served as a loading control. (B) BC-3 cells were transfected with siRNAs specific for Pim-1, -2, -3, or control siRNA (si ctrl), and treated with TPA 48 h after transfection. After another 48 h, whole cell extracts were collected and analyzed by Western blotting with anti-Pim-1, -2, -3, and -K8.1 antibodies. Anti-tubulin served as a loading control. (C) Whole cell extracts from (A) were immunoprecipitated with anti-Pim-1 and -3 or control IgG antibodies and subjected to an in vitro kinase assay towards co-precipitated proteins. The samples were resolved by SDS-PAGE (8%) followed by autoradiography. The kinase filter was immunoblotted with anti-LANA, -Pim-1, and -3 antibodies.

(A) The reporter plasmid pGL3-7XTR (0.1 µg) was co-transfected with expression vectors for LANA (0.5 µg) with and without increasing concentrations of V5-tagged vectors for Pim-1 or -3 (indicated below the graph) into 293 cells. The total transfected DNA concentrations (µg) were kept constant by including an empty control vector. 48 h after transfection, cell extracts were collected for the luciferase assay. (B) Increasing amounts of V5-tagged Pim-1KD or -3KD expression vectors were co-transfected with constant amount of pGL3-7XTR (0.1 µg), LANA (0.5 µg), and V5-Pim-1 or -3 (0.5 µg each) wt vectors into 293 cells. The DNA concentrations (µg) for Pim-1KD and -3KD cDNAs used are indicated below the graph, while the total DNA concentrations (µg) were kept constant by including an empty control vector. Cell extracts were analyzed as in (A). (C) The pGL3-7XTR (0.1 µg), V5-tagged Pim-1 or -3 (0.5 µg each) wt vectors were co-transfected with 0.5 µg of wt LANA or SS205/206RR mutant of LANA into 293 cells. At 48 h after transfection, dual luciferase assays were performed with extracts of transfected cells. An empty pcDNA3 was used as a filler plasmid to keep the total DNA constant in each transfection. Values represent an average of two independent experiments ±SD. (D) The reporter plasmid pGL2-ORF50p (0.1 µg), as well as expression vectors for RTA (0.5 µg) and wt LANA or SS205/206RR mutant of LANA (0.5 µg) were co-transfected with V5-tagged cDNAs for Pim-1 or -3 (indicated below the graph) into 293 cells. The transfected DNA concentrations (µg) were normalized by including an empty control plasmid. 48 h after transfection, cell extracts were collected for the luciferase assay. Values represent an average of two independent experiments ±SD.

In latency, binding of LANA to the LANA binding sites 1 and 2 (LBS 1 and LBS2) at the terminal repeat (TR) region represses transcription from the TR region. LANA can also repress transcription from the RTA promoter and inhibit RTA to autoactivate its own promoter. Upon stimulation by cytokines, TPA or NaB, Pim-1 and -3 are up-regulated, and they interact with and phosphorylate LANA. The phosphorylation by Pim-1 and -3 counteracts the LANA–mediated inhibition of transcription from the TR region and down-modulates autoactivation of the RTA-promoter, leading to viral reactivation.